Antifungal activity of well-defined chito-oligosaccharide preparations against medically relevant yeasts.

Due to their antifungal activity, chitosan and its derivatives have potential to be used for treating yeast infections in humans. However, to be considered for use in human medicine, it is necessary to control and know the chemical composition of the compound, which is not always the case for polymeric chitosans. Here, we analyze the antifungal activity of a soluble and well-defined chito-oligosaccharide (CHOS) with an average polymerization degree (DPn) of 32 and fraction of acetylation (FA) of 0.15 (C32) on 52 medically relevant yeast strains. Minimal inhibitory concentrations (MIC) varied widely among yeast species, strains and isolates (from > 5000 to < 9.77 µg mL-1) and inhibition patterns showed a time- and dose-dependencies. The antifungal activity was predominantly fungicidal and was inversely proportional to the pH, being maximal at pH 4.5, the lowest tested pH. Furthermore, antifungal effects of CHOS fractions with varying average molecular weight indicated that those fractions with an intermediate degree of polymerization, i.e. DP 31 and 54, had the strongest inhibitory effects. Confocal imaging showed that C32 adsorbs to the cell surface, with subsequent cell disruption and accumulation of C32 in the cytoplasm. Thus, C32 has potential to be used as a therapy for fungal infections.

Antibiotic saving effect of combination therapy through synergistic interactions between well-characterized chito-oligosaccharides and commercial antifungals against medically relevant yeasts.

Combination therapies can be a help to overcome resistance to current antifungals in humans. The combined activity of commercial antifungals and soluble and well-defined low molecular weight chitosan with average degrees of polymerization (DPn) of 17-62 (abbreviated C17 -C62) and fraction of acetylation (FA) of 0.15 against medically relevant yeast strains was studied. The minimal inhibitory concentration (MIC) of C32 varied greatly among strains, ranging from > 5000 µg mL-1 (Candida albicans and C. glabrata) to < 4.9 (C. tropicalis). A synergistic effect was observed between C32 and the different antifungals tested for most of the strains. Testing of several CHOS preparations indicated that the highest synergistic effects are obtained for fractions with a DPn in the 30-50 range. Pre-exposure to C32 enhanced the antifungal effect of fluconazole and amphotericin B. A concentration-dependent post-antifungal effect conserved even 24 h after C32 removal was observed. The combination of C32 and commercial antifungals together or as part of a sequential therapy opens new therapeutic perspectives for treating yeast infections in humans.

Hallmarks of processivity in glycoside hydrolases from crystallographic and computational studies of the Serratia marcescens chitinases.

Degradation of recalcitrant polysaccharides in nature is typically accomplished by mixtures of processive and nonprocessive glycoside hydrolases (GHs), which exhibit synergistic activity wherein nonprocessive enzymes provide new sites for productive attachment of processive enzymes. GH processivity is typically attributed to active site geometry, but previous work has demonstrated that processivity can be tuned by point mutations or removal of single loops. To gain additional insights into the differences between processive and nonprocessive enzymes that give rise to their synergistic activities, this study reports the crystal structure of the catalytic domain of the GH family 18 nonprocessive endochitinase, ChiC, from Serratia marcescens. This completes the structural characterization of the co-evolved chitinolytic enzymes from this bacterium and enables structural analysis of their complementary functions. The ChiC catalytic module reveals a shallow substrate-binding cleft that lacks aromatic residues vital for processivity, a calcium-binding site not previously seen in GH18 chitinases, and, importantly, a displaced catalytic acid (Glu-141), suggesting flexibility in the catalytic center. Molecular dynamics simulations of two processive chitinases (ChiA and ChiB), the ChiC catalytic module, and an endochitinase from Lactococcus lactis show that the nonprocessive enzymes have more flexible catalytic machineries and that their bound ligands are more solvated and flexible. These three features, which relate to the more dynamic on-off ligand binding processes associated with nonprocessive action, correlate to experimentally measured differences in processivity of the S. marcescens chitinases. These newly defined hallmarks thus appear to be key dynamic metrics in determining processivity in GH enzymes complementing structural insights.